Roundworm coproantigen detection

ABSTRACT

A composition, device, kit and method for detecting the presence or absence of roundworm in a fecal sample. The composition, device, kit and method of the present invention may be used to confirm the presence or absence of roundworm in a fecal sample from a mammal that may also be infected with one or more of hookworm, whipworm, and heartworm.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/763,592, filedJun. 15, 2007 (now U.S. Pat. No. 7,736,660).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions, devices, kits and methodsfor the detection of roundworm in animals. More particularly, theinvention relates to compositions, devices, kits and methods fordetecting the presence or absence of roundworm in a fecal sample. Evenmore particularly, the present invention relates to antibodycompositions, devices, kits, and methods for detecting the presence orabsence of roundworm antigen in a fecal sample that may also include oneor more of hookworm, whipworm, and heartworm antigen.

2. Description of the Prior Art

Intestinal roundworm infection is common in animals and, if leftuntreated, can cause serious disease and even death. Although it isrelatively easy to diagnosis a roundworm-infected animal as having aparasitic worm (helminth) infection of some type, it is significantlymore difficult to identify roundworm, specifically, as the causativeworm. This is a problem because roundworm infections are best treatedwhen the infected animal's caregiver has knowledge that roundworm is thespecific source of the infection. For example, such knowledge allows thecaregiver to treat the animal with a drug that is optimally potentagainst roundworm, and therefore to avoid using a drug or drug cocktailthat is generally effective against parasitic worm infections, but notoptimally effective against roundworm.

Current methods for diagnosis of roundworm infections primarily involvemicroscopic examination of fecal samples, either directly in fecalsmears or following concentration of ova and parasites by flotation indensity media. Despite this procedure's high adoption, the method hassignificant shortcomings. These microscopic methods are time consuming,require specialized equipment and have low specificity. In addition, theaccuracy of results of these methods is highly dependent upon the skilland expertise of the operator.

Taxonomic distinctions generally also may be made at a molecular levelby determining whether one or more antigens of one or more antibodies toa particular worm species or to a defined group of worm species arepresent in an animal. For example, Hill et al. (Veterinary Parasitology(1997), vol. 68, pp, 91-102) disclose an enzyme-linked immunosorbentassay (ELISA) test for the detection of antibodies specific for whipwormin sera from porcine animals. While the test of Hill et al. does notcrossreact with sera from pigs infected with roundworm or hookworm, ithas not been determined, whether the test crossreacts with sera frompigs infected with heartworm. Similarly, Yamasaki et al. (J. Clin.Microbiology (2000), vol. 38, pp. 1409-1413) disclose an ELISA testutilizing a recombinant roundworm antigen for the detection ofantibodies specific for roundworm in human sera. Whereas the assay ofYamasaki et al. has been shown to not be crossreactive with hookworm orheartworm, it has not been determined whether it crossreacts withwhipworm. Bungiro and Cappello (A. J. Trop. Med. Hyg. (2005), vol. 73,pp. 915-920) disclose an ELISA to detect infection by the hookwormAncylostoma ceylanicum in an experimental hamster model system but ithas not been determined whether their test also crossreacts with one ormore of roundworm, whipworm and heartworm.

Clinicians have shown little interest in using these assays to diagnoseworm-infected animals. One reason why these assays have not been adoptedis that researchers have not demonstrated that any of them are capableof specifically detecting a particular type of worm at the exclusion ofall other major types of worms. For example, no one has yet developed anassay that specifically detects roundworm but that also has been shownto not crossreact with hookworm, whipworm and heartworm. This inabilityto pinpoint an animal's infection to a single source would causeuncertainty in diagnosis, and therefore would likely result in theadministration of suboptimal treatment.

Further, some of these assays only have been shown to be useful fordetecting antigens or antibodies in a serum sample. This is limitingbecause it often is impractical or difficult to obtain a serum samplefrom a sickened animal. For instance, in the case of an uncooperativeanimal, it may be difficult to stabilize the animal for the purpose ofwithdrawing blood, and in the case of a very sick animal, it may beimpractical to transport the animal to a clinician's office for thatsame purpose. Testing for the presence or absence of a particular wormtype therefore is better performed using an animal material that isreadily obtainable and that does not require transportation of theanimal, such as feces. Antigens present in fecal samples are referred toas coproantigens. In the case of the parasitic worm antigens that aresubject of the present invention, coproantigens are worm antigenspresent in a fecal sample of a host animal.

Another limitation inherent to some of these assays is that they involvethe production and purification of a specific recombinant antigen.Specifically, this is limiting because the steps required to produce andpurify such an antigen can be costly and time-consuming.

What is needed therefore are compositions, devices, kits and methods fordetecting the presence or absence of roundworm in a fecal sample. Theneeded compositions, devices, kits and methods further should be able tospecifically detect the presence or absence of roundworm in a fecalsample that contains one or more of hookworm, whipworm, and heartworm.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery of an unexpectedproperty of polyclonal antibodies. Specifically, it was determined thatpolyclonal antibody raised against either whole roundworm extract,roundworm reproductive tract extract, or roundworm intestinal tractextract, can be used to capture and detect the presence or absence ofroundworm coproantigens in a mammal that is infested by one or more ofwhipworm, heartworm and hookworm. This specificity for roundworm issurprising since roundworms, whipworms, heartworms and hookworms all arerelated nematodes, and a polyclonal antibody raised against a wholeextract, roundworm reproductive tract extract, or roundworm intestinaltract extract, of any one of these worms would be expected to crossreactwith one or more of the other worms, host antigens, or other fecalcomponents. The invention includes assay conditions under which theantibodies of the invention can be used to specifically capture anddetect the presence or absence of roundworm coproantigens in a mammalthat may also be infested by one or more of whipworm, heartworm andhookworm.

The invention, in one aspect, is a device for detecting the presence orabsence of roundworm in a fecal sample in a mammal, such as a canine,feline, bovine, or human, for example. The invention further provides adevice for detecting the presence or absence of roundworm in a fecalsample of a mammal that may also be infected with one or more ofhookworm, whipworm, and heartworm. In one aspect of the invention, thedevice includes a solid support, wherein one or more polyclonalantibodies specific for one or more roundworm antigens are immobilizedon the solid support.

In certain aspects of the invention, the device of the inventionincludes a lateral flow immunoassay device. In other aspects of theinvention, the device of the invention includes an ELISA device.

The invention also includes antibodies and antibody compositions. Morespecifically, the invention relates to polyclonal antibodies that arecapable of specifically binding roundworm coproantigen in a mammal thatmay also be infected with one or more of hookworm, whipworm orheartworm. The antibodies of the invention do not substantially bindhookworm, whipworm or heartworm antigen in a fecal sample. The presentinvention further includes methods of producing such antibodies.

The invention also is a method of detecting the presence or absence ofroundworm in a fecal sample. The method includes contacting a fecalsample with the antibodies and capturing and detecting the presence orabsence of roundworm coproantigens in that fecal sample. The detectionstep may include the detection of the presence or absence of anantigen/antibody complex. The method may further involve providing asecond antibody that binds to the antigen of the antigen/antibodycomplex.

The invention further includes assay kits for detecting roundwormcoproantigen in a fecal sample obtained from a mammal. A kit thereforemay include one or more compositions and/or devices of the presentinvention. For example, the kit may include anti-roundworm antibodiesand means for determining binding of the antibodies to roundwormantigens in the sample. In one particular example, such a kit includesthe device having an immobilized anti-roundworm antibody, one or moreantigen capture reagents (e.g., a non-immobilized labeled antigencapture reagent and an immobilized antigen capture reagent) and washreagent, as well as detector reagent and positive and negative controlreagents, if desired or appropriate. Other components such as buffers,controls, and the like, may be included in such test kits. A kit mayfurther include instructions for carrying out one or more methods of thepresent invention, including instructions for using any device of thepresent invention that is included with the kit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a multi-well plate device of the present invention.

FIG. 1B shows a close up of a single well of the plate of FIG. 1A withan example representation of antibodies immobilized thereto.

FIG. 2 shows a graph of optical density (OD) values obtained from caninefecal samples by following a method of the present invention in a firstexperiment.

FIG. 3 shows a graph of OD values obtained from canine fecal samples byfollowing the method of the present invention in a second experiment.

FIG. 4 shows a graph of OD values obtained from canine fecal samples byfollowing the method of the present invention in a third experiment.

FIG. 5 shows a first graph of OD values obtained from feline fecalsamples by following the method of the present invention in a fourthexperiment.

FIG. 6 shows a second graph of OD values obtained from feline fecalsamples by following the method of the present invention in the fourthexperiment.

FIG. 7 shows a first graph of OD values obtained from canine fecalsamples by following the method of the present invention in a fifthexperiment.

FIG. 8 shows a second graph of OD values obtained from canine fecalsamples by following the method of the present invention in the fifthexperiment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention, in one aspect, is a device for the detection ofintestinal roundworm infection in a mammal, such as a canine, feline,bovine, or human, for example. The device is arranged to aid in thedetection of the presence or absence of roundworm coproantigen in afecal sample from a mammal that may also be infected with one or more ofhookworm, whipworm, and heartworm. In one aspect of the invention, thedevice includes a solid support, wherein one or more polyclonalantibodies, raised against whole roundworm extract, roundwormreproductive tract extract, and/or roundworm intestinal tract extract,and specific for one or more roundworm antigens, are immobilized on thesolid support. The solid support can be, for example, a plate or asubstrate in a lateral flow device.

As shown in FIGS. 1A and 1B, the device of the present invention is, forexample, a multi-well plate 10 including a plurality of wells 12. Eachwell 12 provides a solid support 14 for immobilizing thereon apolyclonal antibody 16 specific to roundworm. The plate 10 may be anImmulon 1B 96-well plate, but is not limited thereto. Alternatively, thedevice could be a lateral flow assay such as that described in U.S. Pat.No. 5,726,010.

The polyclonal antibody 16, generally designated “anti-Toxocara pAB”,that is immobilized on the solid support 14 is produced by administeringa whole extract of a species of Toxocara or an extract of a portion,such as all or part of the reproductive tract or all or part of theintestinal tract, of a species of Toxocara to an animal, such as arabbit, for example, collecting serum from that animal and purifying theanti-Toxocara pAB. Anti-Toxocara pAB specifically is immobilized ontothe solid support 14 of the well 12 of the plate 10 by physicaladsorption. Immobilization of anti-Toxocara pAB onto the solid support14 is performed so that anti-Toxocara pAB will not be washed away by anyprocedures that may be performed, and so that the specific binding ofantigens in a fecal sample to anti-Toxocara pAB is unimpeded by thesolid support 14 or other device surface while the method of the presentinvention is being performed. The device 10 of the present invention issuitable for detecting roundworm antigen by the method of the presentinvention, which may include performing an ELISA assay.

The method of the invention may be used to detect one or more roundwormantigens in a sample. The test sample used in the method of theinvention is a fecal sample. The method of invention may be used to testa fecal sample from any mammal, such as a feline, a canine, bovine or ahuman, for example.

The device 10 of the present invention, which includes anti-Toxocara pABimmobilized on the solid support 14, may be used in conjunction with amethod of the present invention to detect roundworm in the fecal sample.Specifically, an active roundworm infection of an animal may bediagnosed by detecting one or more roundworm coproantigens withanti-Toxocara pAB that is immobilized on the solid support 14 of thedevice 10. “Roundworm coproantigens” are any roundworm componentspresent in a fecal sample that can specifically and stably bind toanti-Toxocara pAB. Roundworm coproantigens therefore may be wholeroundworm, roundworm eggs, roundworm fragments, products secreted,excreted or shed from roundworm or a combination thereof.

“Specific for” or “stably binds” means that anti-Toxocara pAB recognizesand binds to the roundworm coproantigen with greater affinity than toother coproantigens (e.g., a coproantigen from a non-roundworm parasiticworm). Binding specificity can be tested using methodology well known inthe art, for example, ELISA or a radioimmunoassay (RIA). In a method ofthe present invention, roundworm antigen is detected by ELISA. Aspecific example of the ELISA method of the present invention follows.Although the present invention is described with respect to a specificELISA method, however, it is to be understood that those of ordinaryskill in the art will recognize that alternative, additional orsubstitute ELISA steps may be used without deviating from the basicgoal.

A method of the present invention is specifically described withreference to two Examples, which together include five Experiments;however, it is not to be construed as being limited thereto.

Example A

The following materials and methods were used to generate data describedin Experiments 1, 2, 3, and 4 described below.

Polyclonal antibody preparation. The polyclonal antibody “anti-ToxocarapAB” (IgG) was raised in rabbit against whole roundworm (Toxocara canis)extract (Antibody Systems Inc., Hurst, Tex.) and purified from serum byusing standard methods. Briefly, an extract of disrupted wholeroundworms was prepared by harvesting roundworms from infected canineanimals, washing them, and resuspending them in solution. Theresuspended worms were then disrupted by tissue homogenization, pelletedby centrifugation, and resuspended in solution. This resuspension wasadministered to rabbit and serum from the immunized rabbits wascollected. Anti-Toxocara pAB was purified from the plasma of theimmunized rabbits by isolating IgG antibody by protein G affinitychromatography.

Infection and anti-helminth treatment of canine and feline animals. Forall four Experiments, parasitic nematode infection was effected byorally administering about 150-300 larvated eggs of either roundworm(Toxocara), hookworm (Ancylostoma canium), or whipworm (Trichurisvulpis) to a healthy canine or feline. (Specifically, Toxocara canis wasthe roundworm that was administered to canine and Toxocara cati was theroundworm that was administered to feline.) For Experiment 2, fecalsamples were collected from canines known to be naturally infected withheartworm (Dirofilaria immitis). Further, for Experiments 3 and 4 only,canines were treated at post-infection day 91 and felines were treatedat post-infection day 56 with Interceptor®, which is an anthelminticagent commercially available from Novartis Animal Health Inc. of Basel,Switzerland, according to the manufacturer's protocol. It is well knownby those of ordinary skill in the art that Interceptor® is effective forthe removal of roundworms, hookworms, whipworms and heartworms fromcanine and feline animals. Infection was confirmed by microscopicobservation of worm eggs in fecal samples obtained from these canine andfeline animals. Canines and felines producing fecal samples that werefound to be free of worm eggs by microscopic examination were consideredto be uninfected.

Canine and feline fecal sample preparation. Canine and feline animalsknown to be free of parasitic worm infection or to be infected with oneof either roundworm, hookworm, whipworm or heartworm provided the sourceof fecal samples. Samples (approximately 1 gram) from fresh, unpreservedcanine or feline fecal samples were suspended in 4 ml of diluentsolution (“diluent solution” is 0.05 M Tris base; 1 mM EDTA; 0.45%Kathon; 16 mg/ml gentamicin sulfate; 0.05% Tween-20; 40% fetal bovineserum; 10% rabbit serum; and 5% mouse serum). The suspension wascentrifuged at 4000 rpm for 20 minutes to produce a first supernatant.The first supernatant was centrifuged at 12000 rpm for 5 minutes toproduce a second supernatant, which is referred to herein as “fecalextract”.

ELISA assays. Purified anti-Toxocara pAB (5 μg/ml; 100 μl/well) wasimmobilized by physical adsorption on Immulon 1B 96-well platesovernight at 4° C. The plates were then blocked with 1% BSA in 0.1M TrispH 7.0 at 4° C. overnight, followed by drying at room temperature.Approximately 100 μl of fecal extract was added to each well and allowedto incubate at room temperature for one hour. The wells were washed fivetimes with a PBS-Tween-20 solution according to standard methods knownto those of ordinary skill in the art. Free anti-Toxocara pAB waslabeled with horseradish peroxidase (HRP) by using the crosslinkersuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) tocreate a conjugate, and 10 μg/ml of this conjugate was added to eachwell of the 96-well plate. Following a 30′ incubation period at roomtemperature, unbound conjugate was washed from the wells usingPBS-Tween-20 solution according to standard methods known to those ofordinary skill in the art. 50 μl TMBLUE® peroxidase substrate (SeraCareLife Sciences, West Bridgewater, Mass.) was then added to each well andthe plates were incubated for 10′ at room temperature. After stoppingeach enzymatic reaction with 0.1% sodium dodecyl sulfate (SDS) followingthe 10′ incubation period, the optical density (OD) value of each wellof the 96-well plate was measured at A650 by standard spectrophotometrictechniques by using an ELISA plate reader. In this arrangement, the ODvalue obtained for any particular well of the 96-well plate was directlyproportional to the amount of specifically bound antigen present in thewell.

Experiment 1

Anti-Toxocara pAB specifically binds roundworm, but does notspecifically bind hookworm or whipworm, in canine fecal samples.

It was a goal of Experiment 1 to determine whether anti-Toxocara pABspecifically binds coproantigen of roundworm, hookworm, and/or whipwormin canines. OD determinations for 20 canine fecal samples obtained inExperiment 1 are shown in FIG. 2. Specifically, these fecal samples wereobtained from five canine animals known to be free of parasitic worminfection (negLCZ5 d62, negRCZ5 d87, negSBY5 d62, negSVY5 d62, andnegTIY5 d62), five canine animals known to be infected with roundworm(round+KWZ5 d62, round+QKZ5 d62, round+RYZ d62, round+SPY5 d69, andround+WHY5 d62), five canine animals known to be infected with hookworm(hook+LEY5 d62, hook+OGY5 d62, hook+RKY5 d62, hook+SKZ5 d62, andhook+SXZ5 d62), and five canine animals known to be infected withwhipworm (whip+KXZ5 d87, whip+REY5 d85, whip+RQZ5 d85, whip+SEZ d85, andwhip+TGZ d85). Fecal samples were obtained on either post-infection day62 (“d62”), day 69 (“d69”), day 85 (“d85”), or day 87 (“d87”). Thespecific post-infection day chosen for each particular canine animal wasbased on the day that worm egg output was at or near peak levels asdetermined by microscopic inspection.

The average OD measured of the uninfected, hookworm-infected, andwhipworm-infected samples were 0.091, 0.099, and 0.172, respectively(the measured OD of each one of these samples was <0.25), indicatingthat anti-Toxocara pAB did not specifically bind antigen in any of thesesamples. Conversely, the average OD of the fecal samples fromroundworm-infected canines was 1.40, which was about eight times higherthan obtained for the whipworm-infected samples, and about 15 timeshigher than obtained for both the uninfected and hookworm-infectedsamples. These data indicate that anti-Toxocara pAB specifically bindsone or more roundworm antigens, but does not specifically bind anyhookworm or whipworm coproantigen.

Experiment 2

Anti-Toxocara pAB does not specifically bind heartworm in canine fecalsamples.

It was a goal of Experiment 2 to determine whether anti-Toxocara pABspecifically binds heartworm coproantigen. OD determinations for 25canine fecal samples obtained in a second experiment are shown in FIG.3. Specifically, FIG. 3 shows data obtained as the result of testingfecal samples from three canine animals known to be free of parasiticworm infection (negILS 11, negILS 12, and negILS 13), seven canineanimals known to be naturally infected with heartworm (negTRS 403,negTRS 404, negTRS 405, negTRS 406, negTRS 583, negTRS 749, and negTRS868), 11 canine animals know to be infected with roundworm (round+ILS10, round+ILS 18, round+ILS 20, round+ILS 22, round+ILS 29, round+ILS32, round+ILS 36, round+ILS 38, round+ILS 41, round+ILS 67, andround+ILS 51), two canine animals known to be infected with hookworm(hook+ILS 9 and hook+ILS 23), and two canine animals known to beinfected with whipworm (whip+ILS 34 and whip+ILS 39).

The average OD of the uninfected, heartworm-infected, hookworm-infected,and whipworm-infected samples were 0.058, 0.061, 0.074, and 0.074,respectively (the measured OD of each one of these samples was 0.101 orless), indicating that anti-Toxocara pAB did not specifically bindantigen in any of these samples. Conversely, the average OD of the fecalsamples from roundworm-infected canines was 0.599, which was about 10times higher than the average OD measured in the uninfected andheartworm-infected samples, and about eight times higher than theaverage OD of the whipworm-infected samples and the hookworm-infectedsamples. In addition to providing further confirmation thatanti-Toxocara pAB specifically binds one or more roundworm antigens, butdoes not specifically bind any hookworm or whipworm coproantigen, thissecond experiment demonstrates that anti-Toxocara pAB does notspecifically bind any heartworm coproantigen.

Experiment 3

Anti-Toxocara pAB detects roundworm in feces from a canine animal onlywhen the animal has an active roundworm infection.

Once it was determined that anti-Toxocara pAB specifically bindsroundworm, but not hookworm, whipworm or heartworm, Experiment 3 wasperformed to determine whether anti-Toxocara pAB detects roundworm onlyat appropriate times (that is, only when the host animal has an activeroundworm infection). Toward this goal, ELISA data was obtained from allof the uninfected canines and roundworm-infected canines described inExperiments 1 and 2. Specifically, data was generated from fecal samplesobtained from all or some of these roundworm-infected animals one dayprior to infection (“−1”), and at days 23, 31, 38, 44, 48, 52, 93, and105 post-infection. Microscopic inspection of these fecal samplesindicated that the samples obtained on days 38, 44, 48, 52, and 93, butnot on days −1, 23, 31, and 105, were substantially infected withroundworm eggs. (The absence of a substantial number of roundworm eggson days 23 and 31 is consistent with the roundworm life cycle incanines. That is, it is well known in the art that orally administeredworm eggs do not manifest in canine fecal material in substantialnumbers until about one month after introduction. Further, it isexpected that the absence of a substantial number of roundworm eggs onday 105 was due to the anthelmintic treatment administered on day 91.)

As shown in FIG. 4, roundworm was detected by the method of the presentinvention only in fecal samples that were microscopically determined tobe substantially infected with roundworm eggs (i.e., only in samplesobtained on days 38, 44, 48, 52, and 93). Specifically, the average ODvalue generated from fecal samples from roundworm-infected canines oneach of days −1, 23, 31, and 105 (which were microscopically determinedto be substantially free of roundworm eggs), was <0.180. The average ODvalue generated from fecal samples from roundworm-infected canines oneach of days 38, 44, 48, 52, and 93 was 1.316, 1.842, 1.896, 2.295,1.104, which represents a range of about a six-fold to about a 12-foldincrease in OD over the egg-free samples.

Experiment 3 therefore indicates that anti-Toxocara pAB specificallybinds roundworm coproantigen only when a host animal has an activeroundworm infection.

Experiment 4

Anti-Toxocara pAB specifically binds roundworm coproantigen in a felinefecal sample, and does so only when the feline from which the sample wasobtained has an active roundworm infection.

It was a goal of Experiment 4 to determine whether anti-Toxocara pABspecifically binds coproantigen of roundworm in felines.

OD values measured for fecal samples obtained from uninfected felinesand roundworm-infected felines are shown in FIGS. 5 and 6, respectively.Specifically, FIG. 5 shows average OD values (and standard deviations)measured using fecal samples from uninfected felines over the course of75 days. (Each OD value shown in FIG. 5 is the average of six OD valuesobtained from the same fecal sample in six separate ELISA reactions.)Data from the same six uninfected felines, which are designated C081-N,C098-N, C118-N, C091-N, C097-N, and C106-N, are shown for days −1 (i.e.,one day before administration of hookworm infection to thehookworm-infected animals) and 54, and for selected days therebetween.Further, data from three of these six felines, C081-N, C098-N andC118-N, also are shown for each of days 60, 68 and 74.

FIG. 6 shows average OD values (and standard deviations) generated usingfecal samples taken from hookworm-infected felines over the course of 78days. (Each OD value shown in FIG. 6 is the average of six OD valuesgenerated from the same fecal sample in six separate ELISA reactions.)Data from the same six feline animals, which are designated C085-R,C087-R, C104-R, C096-R, C100-R and C107-R, are shown for days −1 (i.e.,one day before administration of roundworm infection to the animals) and54, and for selected days therebetween. Further, data from three ofthese six felines, C085-R, C087-R and C104-R, are shown for days 60 and77, and for selected days therebetween.

Referring to FIG. 5, the average of the average OD values measured forthe uninfected felines was 0.059 or less for each of days −1 and 74 andthe selected days therebetween. Referring to FIG. 6, the average of theaverage OD values measured for the roundworm-infected felines was 0.053or less for each of days −1 and 34 and the selected days therebetween.On day 40, the average of the average OD values measured for theroundworm-infected felines was 0.312, which is about 10-fold higher thanwas seen in the uninfected felines for each of days −1 and 74 and theselected days therebetween, and in the roundworm-infected felines atdays −1 and 34 and the selected days therebetween. Further, severalaverage OD values measured for some of the roundworm-infected felines onpost-infection days 42, 46, 48, 50 and 54 were several-fold higher thanwas seen in the uninfected felines for each of days −1 and 74 and theselected days therebetween, and in the roundworm-infected felines atdays −1 and 34 and the selected days therebetween.

These data indicate that anti-Toxocara pAB specifically binds one ormore coproantigen of roundworm. These data further indicate thatanti-Toxocara pAB may be used to determine whether a feline is or is notinfected with roundworm.

It was another goal of Experiment 4 to determine whether anti-ToxocarapAB detects roundworm only when a feline animal has an active roundworminfection.

Microscopic observation of the fecal samples taken from theroundworm-infected canine animals on post-infection day 60 showed thatthe samples were moderately free of roundworm ova, and microscopicobservation of the fecal samples taken from the hookworm-infectedanimals on post-infection days 63, 68, 70, 74 and 77 showed that thesamples were substantially free of roundworm ova. It is expected thatthe moderate reduction in ova on post-infection day 60 and thesubstantial reduction in ova on post-infection days 63, 68, 70, 74 and77 was due to the anthelmintic treatment administered on post-infectionday 56. Referring to FIG. 6, the average of the average OD valuesmeasured for the roundworm-infected felines was consistent with theobserved reduction of ova number in the fecal samples taken from theseanimals. Specifically, the average of the average OD value measured forthese canines on the days following anthelmintic treatment was 0.063(day 60) or less.

These data indicate that anti-Toxocara pAB specifically binds one ormore coproantigen of roundworm. These data further indicate thatanti-Toxocara pAB may be used to determine whether a feline has or doesnot have an active roundworm infection.

Example B

The following materials and methods were used to generate data describedin Experiment 5 described below.

Polyclonal antibody preparation. One preparation of anti-Toxocara pAB(IgG) was raised in rabbits against extracts from roundworm (T. canis)intestine and a second preparation of anti-Toxocara pAB (IgG) was raisedin rabbits against extracts from roundworm (T. canis) reproductiveorgans, and both preparations were purified from serum by using standardmethods. (For clarity, anti-Toxocara pAB raised against intestine ismore specifically referred to as being “anti-TGUT pAB” and anti-ToxocarapAB raised against reproductive organs is more specifically referred toas being “anti-TOVA pAB”.) Briefly, extracts from dissected femaleroundworm intestine or male and female reproductive organs were preparedby harvesting roundworms from infected canine animals, washing them, anddissecting the organs. The organs were ground in liquid nitrogen andsuspended in Hanks Balanced Salt solution having protease inhibitor.This suspension was administered to rabbits and serum from the immunizedrabbits was collected. Anti-TGUT pAB and anti-TOVA pAB were purifiedfrom the plasma of the immunized rabbits by isolating IgG antibody byprotein G affinity chromatography.

Infection and anti-helminth treatment of canine animals. For Experiment5, parasitic nematode infection was effected by orally administeringabout 150-300 larvated eggs of either roundworm (T. canis), hookworm, orwhipworm to a healthy canine. Infection was confirmed by microscopicobservation of worm eggs in fecal samples obtained from these canineanimals. Canines producing fecal samples that were found to be free ofworm eggs by microscopic examination were considered to be uninfected.

ELISA assays. Purified anti-TGUT pAB or anti-TOVA pAB (3-9 μg/ml; 100μl/well) was immobilized by physical adsorption on Immulon 1B 96-wellplates overnight at 4° C. The plates were then blocked with 1% BSA in0.1M Tris pH 7.0 at 4° C. overnight, followed by drying at roomtemperature. Approximately 100 μl of fecal extract (prepared asdescribed above) was added to each well and allowed to incubate at roomtemperature for one hour. The wells were washed five times with aPBS-Tween-20 solution according to standard methods known to those ofordinary skill in the art. Free anti-TGUT pAB or anti-TOVA pAB waslabeled with HRP by using SMCC to create a conjugate, and 3-9 μg/ml ofthis conjugate was added to each well of the 96-well plate. Following a30′ incubation period at room temperature, unbound conjugate was washedfrom the wells using PBS-Tween-20 solution according to standard methodsknown to those of ordinary skill in the art. 50 μl TMBLUE® peroxidasesubstrate (SeraCare Life Sciences, West Bridgewater, Mass.) was thenadded to each well and the plates were incubated for 10′ at roomtemperature. After stopping each enzymatic reaction with 0.1% SDSfollowing the 10′ incubation period, the OD value of each well of the96-well plate was measured at A650 by standard spectrophotometrictechniques by using an ELISA plate reader. In this arrangement, the ODvalue obtained for any particular well of the 96-well plate was directlyproportional to the amount of specifically bound antigen present in thewell.

Experiment 5

Each of anti-TGUT pAB and anti-TOVA pAB specifically bind roundworm, butneither specifically binds hookworm or whipworm, in canine fecalsamples.

It was a goal of Experiment 5 to determine whether antibody raisedagainst roundworm intestinal tract, specifically referred to as“anti-TGUT pAB”, and/or antibody raised against roundworm reproductivetract, specifically referred to as “anti-TOVA pAB”, specifically bindscoproantigen of roundworm, hookworm, and/or whipworm. OD determinationsfor 16 canine fecal samples obtained in Experiment 5 are shown in eachof FIGS. 7 and 8. (FIG. 7 shows OD values obtained using anti-TGUT pABand FIG. 8 shows OD values obtained using anti-TOVA pAB.) Specifically,these fecal samples were obtained from four canine animals known to befree of parasitic worm infection (negTIY5 d62, negSVY5 d62, negSBY5 d62,and negLCZ5 d62), four canine animals known to be infected withroundworm (round+QKZ5 d62, round+SPY5 d62, round+RYZ d69, and round+WHY5d69), four canine animals known to be infected with hookworm (hook+LEY5d76, hook+OGY5 d76, hook+SXZ5 d84, and hook+SKZ5 d69), and four canineanimals known to be infected with whipworm (whip+SEZ d62, whip+KXZ5 d69,whip+RQZ5 d62, and whip+REY5 d62). Fecal samples were obtained on eitherpost-infection day 62 (“d62”), day 69 (“d69”), day 76 (“d76”), or day 84(“d84”). The specific post-infection day chosen for each particularcanine animal was based on the day that worm egg output was at or nearpeak levels as determined by microscopic inspection.

Referring specifically to FIG. 7, for anti-TOUT pAB, the average ODmeasured of the uninfected, hookworm-infected, and whipworm-infectedsamples were 0.075, 0.083, and 0.082, respectively (the measured OD ofeach one of these samples was <0.096), indicating that anti-TGUT pAB didnot specifically bind antigen in any of these samples. Conversely, theaverage OD of the fecal samples from roundworm-infected canines was0.385, which was about 4.5 to 5 times higher than obtained for theuninfected, hookworm-infected and whipworm-infected samples. These dataindicate that anti-TGUT pAB specifically binds one or more roundwormantigens, but does not specifically bind any hookworm or whipwormcoproantigen.

Referring specifically to FIG. 8, for anti-TOVA pAB, the average ODmeasured of the uninfected, hookworm-infected, and whipworm-infectedsamples were 0.588, 0.820, and 0.590, respectively (the measured OD ofeach one of these samples was <0.916). Conversely, the average OD of thefecal samples from roundworm-infected canines was 3.244, which was about4 to 5.5 times higher than obtained for the uninfected,hookworm-infected and whipworm-infected samples. These data indicatethat anti-TGUT pAB specifically binds one or more roundworm antigens,but does not specifically bind any hookworm or whipworm coproantigen.

While the composition, device and method of the present invention havebeen described with respect to a specific embodiment and specificExamples, it is to be understood that variations to the device and/orthe method of the present invention may be made without departing fromthe spirit and scope of the invention. For example, it is to beunderstood that a polyclonal antibody other than anti-Toxocara pAB, ormonoclonal antibodies, single chain antibodies (scFv), chimericantibodies, or fragments of an antibody may be substituted foranti-Toxocara pAB. Another polyclonal antibody that is specific forroundworm could be prepared, for example, by administering one or moreantigens specific for roundworm to an animal. Further, whereasanti-Toxocara pAB was raised in rabbit, polyclonal antibody also may beraised in, for example, a human or other primate, mouse, rat, guineapig, goat, pig, cow, sheep, donkey, dog, cat, chicken, or horse. Anantibody used in the device of the invention also may be any antibodyclass, including for example, IgG, IgM, IgA, IgD and IgE. Means forpreparing and characterizing antibodies are well know in the art. See,e.g., Dean, Methods Mol. Biol. 80:23-37 (1998); Dean, Methods Mal. Biol.32:361-79 (1994); Bailegi, Methods Mol. Biol. 32:381-88 (1994); Gullick,Methods Mol. Biol. 32:389-99 (1994); Drenckhahn et al. Methods Cell.Biol. 37:7-56 (1993); Morrison, Ann. Rev. Immunol. 10:239-65 (1992);Wright et al. Crit. Rev. Immunol. 12:125-68 (1992).

An antibody used in the devices, methods and kits of the invention canalso be a single chain antibody (scFv), or an antigen binding fragmentof an antibody. Antigen-binding fragments of antibodies are a portion ofan intact antibody comprising the antigen binding site or variableregion of an intact antibody, wherein the portion is free of theconstant heavy chain domains of the Fc region of the intact antibody.Examples of antibody fragments include Fab, Fab′, Fab′-SH, F(ab′)₂ andF, fragments.

Antibodies used in the device of the invention may be immobilized on thesolid support by any methodology known in the art, including, forexample, covalently or non-covalently, directly or indirectly, attachingthe antibodies to the solid support. Therefore, while anti-Toxocara pABis attached to the solid support by physical adsorption (i.e., withoutthe use of chemical linkers) in the embodiment described, it iscontemplated that anti-Toxocara pAB or other antibodies may beimmobilized to the solid support by any chemical binding (i.e., with theuse of chemical linkers) method readily known to one of skill in theart.

The solid support of the device is not limited to being a polystyrene96-well plate. The solid support may be any suitable material for theimmobilization of antibodies specific for roundworm. For example, thesolid support may be beads, particles, tubes, wells, probes, dipsticks,pipette tips, slides, fibers, membranes, papers, natural and modifiedcelluloses, polyacrylamides, agaroses, glass, polypropylene,polyethylene, dextran, nylon, amylases, plastics, magnetite or any othersuitable material readily known to one of skill in the art.

The device of the present invention also is not limited to being adevice that is suitable for performing an ELISA assay. For example, thedevice may be one that is suitable for performing a lateral flow assay.An exemplary device that is useful for performing a lateral flow assaythat is useful in the present invention is described in U.S. Pat. No.5,726,010, which is incorporated herein by reference in its entirety.The device for performing a lateral flow assay therefore may be a SNAP®device, which is commercially available from IDEXX Laboratories, Inc. ofWestbrook, Me.

The device may optionally include one or more labeled antigen capturereagents that may be mixed with a fecal sample prior to application to adevice of the invention. When the labeled antigen reagent is included,the labeled antigen capture reagent may or may not be deposited or driedon the solid surface of the device. By “antigen capture” is meant anycompound that is specific for the antigen of interest. The labeledantigen capture reagent, whether added to a fecal sample orpre-deposited on the device, may be, for example, a labeled antibodyspecific for a roundworm antigen. For example, a roundworm-specificantibody conjugated with horseradish peroxidase may be used as a labeledantigen capture reagent.

The device also may optionally include a liquid reagent that transports,such as when the device includes the SNAP® device, for example, orfacilitates removal of, such as when the device includes a 96-wellplate, for example, unbound material (e.g., unreacted fecal sample andunbound antigen capture reagent) away from the reaction zone (solidphase). The liquid reagent may be a wash reagent and serve only toremove unbound material from the reaction zone, or it may include adetector reagent and serve to both remove unbound material andfacilitate antigen detection. For example, in the case of an antigencapture reagent conjugated to an enzyme, the detector reagent includes asubstrate that produces a detectable signal upon reaction with theenzyme-antibody conjugate at the reaction zone (solid phase).Alternatively, in the case of a labeled antigen capture reagentconjugated to a radioactive, fluorescent, or light-absorbing molecule,the detector reagent acts merely as a wash solution facilitatingdetection of complex formation at the reactive zone by washing awayunbound labeled reagent.

The liquid reagent may further include a limited quantity of an“inhibitor”, i.e., a substance that blocks the development of thedetectable end product. A limited quantity is defined as being an amountof inhibitor sufficient to block end product development until most orall excess, unbound material is transported away from the second region,at which time detectable end product is produced.

The device of the present invention may also include various bindingreagents immobilized at locations distinct from the antigen capturereagent(s). For example, an immunoreagent (an antibody, antigen orpeptide) that recognizes a species-specific (e.g., worm-specific)antibody portion of a labeled antibody or antigen capture reagent or anenzyme portion of an enzyme-labeled reagent can be included as apositive control to assess the viability of the reagents within thedevice. For example, a positive control may be an anti-horseradishperoxidase antibody that has been raised in, for example, a goat or amouse. Additionally, a reagent, e.g., an antibody, isolated from anon-immune member of the species from which the antibody portion of theantigen-antibody complex was derived can be included as a negativecontrol to assess the specificity of immunocomplex (i.e.,antigen-antibody complex) formation.

In addition to being designed to detect roundworm in a fecal sample, thedevice of the invention optionally may be designed to allow one or moreother diagnostic tests to be performed. For example, the solid supportmay also include reagents for the detection of one or more non-roundwormworm parasites, one or more non-worm parasites, one or more viruses, orone or more bacteria. The reagents for the detection of one or morenon-roundworm worm parasites, one or more non-worm parasites, one ormore viruses or one or more bacteria may be, for example, one or moreantibodies or one or more antigens recognized by antibodies specific forone or more non-roundworm worm parasites, one or more non-wormparasites, one or more viruses or one or more bacteria.

In the methods of the present invention, detection of a roundworminfection is accomplished by detecting the presence or absence of one ormore roundworm antigens in a fecal sample. The soluble portion of afecal sample to be tested may be collected by any protocol known in art.For example, in addition to the specific protocol described above, thesoluble portions of the sample may be collected using filtration,centrifugation, or simple mixing followed by gravimetric settling.

The methods include contacting the fecal sample with one or moreantibodies specific for one or more roundworm antigens under conditionsthat allow an antigen/antibody complex, i.e., an immunocomplex, to form.That is, an antibody specifically binds to a roundworm antigen presentin the fecal sample. One of skill in the art would be familiar withassays and conditions that are used to detect antigen/antibody complexbinding. For example, the antigen/antibody complex may be detected usinga secondary antibody that binds to the antigen/antibody complex. Theformation of a complex between roundworm antigen and anti-roundwormantibodies in the sample may be detected using any suitable method knownin the art. Further, the amount of antibody-antigen complexes can bedetermined by methodology known in the art. A level that is higher thanthat formed in a control sample indicates a roundworm infection.

Alternative steps of the method of the present invention may includeapplying the fecal sample to a device of the invention, which includesan immobilized antibody specific for roundworm antigen, and detectingthe presence or absence of the roundworm antigen in the fecal sample.Antibodies specific for antigens of roundworms may be directly orindirectly attached to a solid support or a substrate such as amicrotiter well, magnetic bead, non-magnetic bead, column, matrix,membrane, fibrous mat composed of synthetic or natural fibers (e.g.,glass or cellulose-based materials or thermoplastic polymers, such as,polyethylene, polypropylene, or polyester), sintered structure composedof particulate materials (e.g., glass or various thermoplasticpolymers), or cast membrane film composed of nitrocellulose, nylon,polysulfone or the like (generally synthetic in nature). All of thesesubstrate materials may be used in suitable shapes, such as films,sheets, or plates, or they may be coated onto or bonded or laminated toappropriate inert carriers, such as paper, glass, plastic films, orfabrics. Suitable methods for immobilizing peptides on solid phasesinclude ionic, hydrophobic, covalent interactions and the like.

The method of the present invention need not include the use of solidphases or substrates, however. For example, the methods may includeimmunoprecipitation methods which do not require the use of solid phasesor substrates.

In some embodiments of the invention, the antigen/antibody complex isdetected when an indicator reagent, such as an enzyme conjugate, whichis bound to the antibody, catalyzes a detectable reaction. Optionally,an indicator reagent including a signal generating compound may beapplied to the antigen/antibody complex under conditions that allowformation of a detectable antigen/antibody/indicator complex.Optionally, the antibody may be labeled with an indicator reagent priorto the formation of an antigen/antibody complex.

The formation of an antigen/antibody complex or anantigen/antibody/indicator complex in some of the methods of the presentinvention specifically may be detected by radiometric, calorimetric,fluorometric, size-separation, or precipitation methods. Detection of anantigen/antibody complex also may be accomplished by the addition of asecondary antibody that is coupled to an indicator reagent including asignal generating compound. Indicator reagents including signalgenerating compounds (labels) associated with a polypeptide/antibodycomplex may be detected using the methods described above and mayinclude chromogenic agents, catalysts such as enzyme conjugates,fluorescent compounds such as fluorescein and rhodamine,chemiluminescent compounds such as dioxetanes, acridiniums,phenanthridiniums, ruthenium, and luminol, radioactive elements, directvisual labels, as well as cofactors, inhibitors, magnetic particles, andthe like. Examples of enzyme conjugates include alkaline phosphatase,horseradish peroxidase, beta-galactosidase, and the like. The selectionof a particular label is not critical, but it will be capable ofproducing a signal either by itself or in conjunction with one or moreadditional substances.

Antibodies, including secondary antibodies, may be labeled with any typeof label known in the art, including, for example, fluorescent,chemiluminescent, radioactive, enzymes, colloidal particles,radioisotopes and bioluminescent labels. In various embodiments of theinvention, the one or more of the antibodies of the invention arelabeled with an enzyme, a colloidal particle, a radionuclide or afluorophor. The particulate label can be, for example, a colored latexparticle, dye sol, or gold sol conjugated to an antibody specific for aroundworm antigen.

Methods of the invention include, but are not limited to those based oncompetition, direct reaction or sandwich-type assays, including, but notlimited to ELISA, RIA, immuno-fluorescent assays (IFA), hemagglutination(HA), fluorescence polarization immunoassay (FPIA), and microtiter plateassays (any assay done in one or more wells of a microtiter plate). Oneassay of the invention includes a reversible flow chromatographicbinding assay, which may be performed, for example, by using a SNAP®device. See U.S. Pat. No. 5,726,010.

Methods of the invention facilitate sandwich or competition-typespecific binding assays. In a sandwich assay, antigen capture reagentsare immobilized in a reactive zone. These antigen capture reagents mayspecifically bind to antigens in the fecal sample being tested.Specifically, these antigen capture reagents specifically bind to anantigen from a roundworm, if present in the fecal sample. Followingbinding of the antigen from the sample, the antigen capturereagent/antigen complex is detected by any suitable method. For example,the complex may be reacted with labeled specific binding reagents (e.g.,an enzyme-antibody conjugate) and antigen detected (e.g., upon reactionwith substrate).

In other embodiments of the method of the present invention, acompetition assay is performed. In a competition assay, antigen capturereagents are immobilized at the reactive zone and are contactedsimultaneously with antigen from a sample and labeled antigen (e.g., anantigen-enzyme conjugate). The amount of label detected at the reactivezone is inversely proportional to the amount of antigen in the sample.

In some embodiments of the invention, antibodies specific for aroundworm antigen or antigens are attached to a solid phase orsubstrate. The fecal sample potentially including an antigen fromroundworm is added to the substrate. Antibodies that specifically bindroundworm are added. The antibodies may be the same antibodies used onthe solid phase or they may be from a different source or species.Further, these antibodies may be linked to an indicator reagent, such asan enzyme conjugate. Wash steps may be performed prior to each addition.A chromophore or enzyme substrate may be added and color may be allowedto develop. The color reaction may be stopped and the color may bequantified using, for example, a spectrophotometer.

In other embodiments of the invention, antibodies specific for aroundworm antigen or antigens are attached to a solid phase orsubstrate. A fecal sample potentially including a roundworm antigen isadded to the substrate. Second anti-species antibodies that specificallybind antigens of roundworms are added. These second antibodies are froma different species than are the solid phase antibodies. Thirdanti-species antibodies that specifically bind the second antibodies andthat do not specifically bind the solid phase antibodies are added. Thethird antibodies may include an indicator reagent, such as an enzymeconjugate. Wash steps may be performed prior to each addition. Achromophore or enzyme substrate may added and color may be allowed todevelop. The color reaction may be stopped and the color may bequantified using, for example, a spectrophotometer.

In a specific example, the method of the present invention is performedin conjunction with a device that is a lateral flow assay device byadding a prepared fecal sample to a flow matrix of the device at a firstregion (a sample application zone). The prepared fecal sample is carriedin a fluid flow path by capillary action to a second region of the flowmatrix where a particulate label capable of binding and forming a firstcomplex with an antigen in the fecal sample. The particulate label canbe, e.g., a colored latex particle, dye sol, or gold sol conjugated toan antibody specific for a roundworm antigen. The first complex iscarried to a third region of the flow matrix where an antibody thatspecifically binds a roundworm antigen is immobilized at a distinctlocation. A second complex is formed between the immobilized antibodyand the first complex. The particulate label that is part of the secondcomplex can be directly visualized.

Roundworm antibody may be an immobilized antigen capture reagent in areaction zone (solid phase). A second antigen capture reagent, i.e., asecond roundworm antibody that has been conjugated to a label, eithermay be added to the sample before the sample is added to the device, orthe second antigen capture reagent can be incorporated into the device.For example, the labeled antigen capture reagent may be deposited anddried on a fluid flow path that provides fluid communication between asample application zone and the solid phase. Contact of the labeledantigen capture reagent with the test sample can result in dissolutionof the labeled antigen capture reagent.

The invention further includes assay kits (e.g., articles ofmanufacture) for detecting roundworm in a fecal sample. A kit thereforemay include one or more devices of the present invention. For example,the kit may include anti-roundworm antibodies and means for determiningbinding of the antibodies to roundworm antigens in the sample. In oneparticular example, such a kit includes the device having an immobilizedanti-roundworm antibody, one or more antigen capture reagents (e.g., anon-immobilized labeled antigen capture reagent and an immobilizedantigen capture reagent) and wash reagent, as well as detector reagentand positive and negative control reagents, if desired or appropriate.Other components such as buffers, controls, and the like, known to thoseof ordinary skill in art, may be included in such test kits. Therelative amounts of the various reagents can be varied, to provide forconcentrations in solution of the reagents that substantially optimizethe sensitivity of the assay. Particularly, the reagents can be providedas dry powders, usually lyophilized, which on dissolution will providefor a reagent solution having the appropriate concentrations forcombining with a sample. The antibodies, assays, and kits of theinvention are useful, for example, in the diagnosis of individual casesof roundworm infection in a patient, as well as epidemiological studiesof roundworm outbreaks. The kit may further include instructions forcarrying out one or more methods of the present invention, includinginstructions for using any device of the present invention that isincluded with the kit.

The methods of the invention for detection of roundworm infection can becombined with other diagnostic assays to detect the presence of otherorganisms or conditions. For example, assays of the invention can becombined with reagents that detect one or more non-roundworm worm fecalparasite, one or more non-worm fecal parasite, one or more virus, one ormore bacteria, one or more blood-borne parasites or occult blood or acombination thereof. By providing two or more unique binding sites in asingle assay device (such as, for example, two unique spots on a SNAP®assay device), the present invention allows for detection of two or moreorganisms from a single sample. In one embodiment, there are threeunique spots for detection of past or present infection from threeorganisms (the spots being either antigen or antibody binding reagents)from a single sample (i.e. the same, single sample is presented to thethree capture reagents on a single device).

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms, while retaining their ordinary meanings. The terms andexpressions which have been employed are used as terms of descriptionand not of limitation, and there is no intention that in the use of suchterms and expressions of excluding any equivalents of the features shownand described or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments, optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the description and the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

A number of examples to help illustrate the invention have beendescribed. Nevertheless, it will be understood that variousmodifications may be made without departing from the spirit and scope ofthe invention. Accordingly, other embodiments are within the scope ofthe claims appended hereto.

1. A device for detecting the presence or absence of Toxocara roundwormantigen in a fecal preparation from a mammal, comprising a solidsupport, wherein one or more antibodies specific for one or moreToxocara roundworm antigens are immobilized on the solid support.
 2. Thedevice of claim 1 wherein the one or more antibodies specific for one ormore Toxocara roundworm antigens are not specific for any coproantigenpresent in the fecal preparation selected from the group consisting ofhookworm, whipworm, and heartworm.
 3. The device of claim 1 wherein theone or more antibodies are specific for an extract of whole Toxocararoundworm, an extract of Toxocara roundworm intestine or an extract ofToxocara roundworm reproductive organ.
 4. The device of claim 3 whereinthe Toxocara roundworm is Toxocara canis.
 5. The device of claim 1wherein one or more of the one or more antibodies are specifically boundto one or more Toxocara roundworm coproantigens.
 6. The device of claim1 wherein one or more of the one or more antibodies are labeled.
 7. Thedevice of claim 1 wherein the device is an enzyme-linked immunosorbentassay device.
 8. The device of claim 7 wherein the enzyme-linkedimmunosorbent assay device is a lateral flow immunoassay device.
 9. Thedevice of claim 1 wherein the mammal is a canine or a feline.
 10. Thedevice of claim 1 wherein the device further includes one or morereagents for the detection of one or more of the group consisting of:one or more non-roundworm worm parasites, one or more non-wormparasites, one or more viruses, and one or more bacteria, said one ormore reagents selected from the group consisting of one or moreantibodies or one or more antigens recognized by antibodies specific forone or more non-roundworm parasites, one or more non-worm parasites, oneor more viruses and one or more bacteria.
 11. The device of claim 1,wherein the one or more Toxocara roundworm antigens is Toxocara canisantigen or Toxocara cati antigen.
 12. A device for determining thepresence or absence of an intestinal Toxocara roundworm infection in amammal, the device comprising a solid support, wherein one or moreantibodies specific for one or more Toxocara roundworm coproantigens areimmobilized on the solid support.
 13. The device of claim 12 wherein theone or more antibodies specific for one or more Toxocara roundwormcoproantigens are not specific for any coproantigen from the mammalselected from the group consisting of hookworm, whipworm, and heartworm.14. The device of claim 12 wherein the one or more antibodies arespecific for an extract of whole Toxocara roundworm, an extract ofToxocara roundworm intestine or an extract of Toxocara roundwormreproductive organ.
 15. The device of claim 12 wherein the Toxocararoundworm is Toxocara canis.
 16. The device of claim 12 wherein one ormore of the one or more antibodies are specifically bound to one or moreToxocara roundworm coproantigens.
 17. The device of claim 12 wherein oneor more of the one or more antibodies are labeled.
 18. The device ofclaim 12 wherein the device is an enzyme-linked immunosorbent assaydevice.
 19. The device of claim 18 wherein the enzyme-linkedimmunosorbent assay device is a lateral flow immunoassay device.
 20. Thedevice of claim 12 wherein the mammal is a canine or a feline.
 21. Thedevice of claim 12, wherein the one or more Toxicara roundwormcoproantigens are Toxocara canis or Toxocara cati coproantigens.
 22. Akit for detection of one or more Toxocara roundworm antigens in a sampleof a mammal, the kit comprising the device of any one of claims 1-11 and12-21, and one or more reagents sufficient for the detection of the oneor more Toxocara roundworm antigens.
 23. The kit of claim 22 wherein theone or more reagents are selected from the group consisting of one ormore indicator reagents, one or more antibody labeling compounds, one ormore enzyme substrates, one or more antibodies specific for one or moreToxocara roundworm antigens, one or more anti-species antibodies, one ormore antigen capture reagents, one or more inhibitors, and one or morewash reagents.
 24. The kit of claim 22 wherein the one or more Toxocararoundworm antigens are Toxocara roundworm coproantigens.
 25. The deviceof claim 1 wherein the one or more Toxocara roundworm antigens areToxocara roundworm coproantigens.